Interaction of Tim23 to micelles (DHPC)

3.4.2.1 Chemical Shift perturbation of Tim23ims with the detergent DHPC and its micelles

The presence of the slow exchange regime of Tim23-membrane interaction at NMR timescale where resonances from the bound form were not visible in NMR spectra hampered the structural characterization of the membrane bound form of Tim23ims. Therefore, we have used another membrane mimic (the DHPC detergent and its micelles) to structurally characterize the Tim23ims-membrane interaction.

Upon titration of Tim23ims with increasing amount of DHPC, changes in the chemical shift of backbone resonances of Tim23ims on binding to the DHPC molecules along with the changes in the resonance intensities using ¹⁵N-¹H-HSQC spectra were monitored. The addition of DHPC below its critical micellar concentration (CMC) (i.e. up to 16 mM) did not induce any changes in the Tim23ims spectra.

However, the further stepwise addition of DHPC above its CMC, where micelles are formed (i.e. at 24mM and 32mM) caused progressive changes in the backbone amide resonances of Tim23ims for the residues involved in binding to DHPC micelles (Figure 37, A). The observance of chemical shift changes along with small, changes in resonance intensities (at 24mM and 32mM DHPC concentration) is a characteristic of presence of fast to intermediate exchange at NMR time scale. The observed changes in intensity can be attributed to the structural flexibility of the complex or to the chemical exchange (between the DHPC micellar bound and free form of Tim23ims)

Based on the degree of the chemical shift changes in Tim23ims upon micellar (DHPC) binding, the residues can be grouped into four categories: (1) the most perturbed residues 1-7, (2) the intermediately perturbed residues 33-46 and (3) the less perturbed 53-62 and (4) the least perturbed residues or non-interacting residue (7-33, 61-93) (Figure 37 B). The similar pattern of reduction in the resonance intensities was found in the aforementioned regions. The micellar binding regions of Tim23 are similar to the liposome binding region identified in section 3.4.1.

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Figure 37:Tim23 interacts with DHPC micelles. (A) Superposition of 2D ¹H-¹⁵N HSQC spectra of Tim23 without DHPC (black), with 8mM DHPC (yellow), 16mM DHPC (purple) 24mM DHPC and 32mM DHPC (red). The progressive changes in the resonance positions of L4 and F5 are indicated by arrows. (B) Average chemical shift perturbation (CSP) of Tim23 resonances upon addition of 24mM DHPC (green) and 32mM DHPC (red). The grey line represents maximum CSP value of the C- terminal 50 residues.

3.4.2.2 ¹H-¹⁵N HetNOE of micelle-bound Tim23ims

In order to probe the backbone dynamics of micellar bound Tim23ims in detail, we measured the steady state ¹H-¹⁵N HetNOE of Tim23ims in presence of micelles (saturating amounts of 50mM DHPC). On average, positive ¹H-¹⁵N HetNOE value of 0.42 was obtained for an N-terminal membrane binding site (1-7) of Tim23ims in comparison of negative ¹H-¹⁵N HetNOE value observed in the free form (Figure 38).

This observed change in the ¹H-¹⁵N HetNOE values is a direct evidence for the increase in rigidity of HN bond vector for these residues upon binding to DHPC micelles.

However, the magnitude of ¹H-¹⁵N HetNOE values of the micellar bound Tim23ims is still less than as would be expected in for the presence of a rigid secondary structure in proteins (0.6-0.8). The residues 91-94 also show the increase in ¹H-¹⁵N HetNOE value in comparison to free Tim23ims but negative sign is indicative for flexible C-terminus of Tim23 even in micellar bound form.

For the intermediately perturbed residues, the less significant, small positive changes in ¹H-¹⁵N HetNOE values were observed suggesting them to be disordered in the micellar bound form. Moreover, the lack of overall change in ¹H-¹⁵N HetNOE

3.4 Interaction studies of Tim23 with mitochondrial membrane mimics 107

values of micellar bound Tim23ims, suggests that non-interacting residues retains a similar disordered state in the micellar bound form.

The chemical shift perturbations and ¹H-¹⁵N HetNOE data together suggests two regions i.e. N terminal (1-7) and intermediate (31-46) of Tim23ims are involved in binding to the membrane mimicking micelles (DHPC) but remain dynamic and exchange between the in the micelle bound and free form of Tim23ims.

Figure 38: ¹H-¹⁵N-HetNOE intensity ratios (I_sat/I_ref=I/I_o) for Tim23ims in the presence (50mM) and the absence (grey) of DHPC micelles. The error bars are calculated based on the observed S/N ratio.

3.4.2.3 Structural analysis of micelle bound Tim23

In order to understand the change in the conformation of Tim23ims upon micelle binding (50mM DHPC), we characterized the changes in its backbone dihedral angles by evaluating the deviations of the measured C^α chemical shift from the sequence based random coil values i.e. chemical shift index (CSI ) of C^α in micelle-bound Tim23 form (50mM DHPC). The average CSI value for all residues remained below ±0.5ppm

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(except for Ser 2) indicating lack of rigid secondary structural elements present in Tim23ims even upon binding to DHPC micelles. In the main N-terminal micelle binding site (involving residues 1-7) only three residues 2, 3, 4 contain continuous stretch of positive values. Additionally in second micellar binding region of Tim23ims (involving residues 31-46), positive values were seen at stretch for residues 40-44. This precludes the existence of secondary structural elements (α-helix or β-sheet) in micellar bound Tim23ims.

Figure 39: Micelle bound Tim23ims lacks rigid secondary structure: Deviations of the experimentally measured C^αchemical shift from that of random coil values is plotted as a function of Tim23ims sequence.

For clarity only micellar binding regions present in first 50 residues of Tim23 ims are shown.

3.4.2.4 Intermolecular NOE based structure of micelle bound Tim23 ^1-13

Further structural aspects of micellar bound Tim23 were investigated using the ¹H-¹H NOESY of most perturbed N-terminal membrane binding region of Tim23 (1-13) in DHPC micelles (200mM). The intermolecular NOEs were observed between the hydrophobic side chains of DHPC detergent and side chain ¹H for the W3 and F5. The presence of sequential H^N-H^N NOE's among residues Trp³, Leu⁴, Phe⁵ and Gly⁶ indicates that these residues are presumably involved in the formation of a turn. The absence of medium and long range NOE's for the residues 7-13 resulted in these residues being unstructured. To gain the insights into the binding of these residues in micellar bound form of Tim23 (1-13), the structure of the micellar bound Tim23 (1-13) peptide was calculated using 70 short range, 4 medium range and 2 long range NOEs.

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Furthermore, the ensemble of 15 low energy structures calculated showed poor convergence for the overall molecule. However, when the residues 2-6 are superimposed, the backbone and side-chain r.m.s.d were found to be 0.33±0.13 Å and 0.59+/-0.21 Å, respectively, indicating that residues 2-6 adopts a well ordered structure (Figure 40, B and C). On the other hand, when the residues 7-13 are superposed, the backbone and side-chain r.m.s.d was found to be 2.20±0.52 Å and 3.36±0.68 Å respectively. Most importantly, the side-chains of residues Trp³, Leu⁴ and Phe⁵ are oriented in same direction (Figure 40 C, D, and E). These side chains of Trp³, Leu⁴ and Phe⁵ forms a hydrophobic cluster and are involved in the interaction with the hydrophobic core of micelles DHPC.

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Figure 40: Structural analysis of micellar bound N–terminal (1-13) peptide of Tim23.(A) Intermolecular NOEs between side chain HE1 of Trp³ and the hydrophobic tails of protonated DHPC are highlighted with asteriks 1, 2, 3 as in (G) and interresidual side chain ¹H NOEs between side chain HE1 of Trp³ with side chain of L4HB and HD1HD2 are labeled. (B) The ¹H-¹H NOE based structure of micellar Tim23(1-13) showing the good convergence of 15 conformers low energy for the N terminal residues 2-6. (C) Enlarged view of ensemble of 15 low energy structures higlighting hydrophobic cluster invoving side chains of residues W3,L4 and F5 that pointing outward. (D) The hydrophobic cluster for lowest energy conformer was shown for clarity with residues of hydrophobic cluster marked in red. (E) Surface represenation of Tim23 peptide 1-13 with hydrophobic cluster marked in yellow. (F) Histogram for number of NOE restrains incorporated for structure calculation:white, grey and black bars represents short, medium and long range restraints. (G) Chemical structure of DHPC. CHn groups of the hydrophobic tails. The signals arising from the hydrophobic end (−CH₃ marked as 1 and antepenultimate

−CH2 as 2 and others −CH2 as 3 except two unmarked. These signals are marked in (A) as labels+

asteriks.

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